Depletion of oxygen (O2) levels and reduction in the ATP synthesis (or even its complete blockage) are important characteristics of mitochondrial dysfunction; features that are often correlated with neurodegeneration. The measurement of oxygen consumption rate (OCR) is thus essential to evaluate cellular metabolism, survival, and neuroprotective strategies. In the present chapter, we describe the oxygen consumption assay using a Clark-type oxygen electrode in different types of samples named cells suspension (from primary and established cell culture), brain slices (ex vivo), and fresh brain tissues. In addition, we demonstrate herein how the program Oxygraph can be used in order to analyze the data and different approaches to normalize it.3D Cell culture is an alternative to animal use in many drug development and toxicity studies. The 3D cell culture can mimic and reproduce the original tissue microenvironment, morphology, and mechanical and physiological characteristics, to provide a more realistic and reliable response as compared to two-dimensional cultures. 3D cell culture encapsulated in alginate beads is a very simple and relatively inexpensive tool that is easy to handle and to maintain. The alginate beads function as a scaffold that imprisons cells and allows 3D cell growth, to generate spheroids that can have greater genic expression and cell-cell communication as a nano or microtissue. The HepG2 cell line is a human hepatocellular carcinoma cell derivative. HepG2 cells preserve several of the characteristics of hepatocytes and are therefore often used in toxicity studies. Here, we describe HepG2 cell encapsulation in alginate beads and analyze the resulting spheroids formed within the alginate beads by immunocytochemistry, by staining a certain structure with a specific antibody coupled with a fluorophore. This method preserves the beads and enables cell analysis by confocal microscopy.Historically, the ocular toxicity of manufactured consumer materials has been evaluated using the rabbit in vivo Draize rabbit eye test. The animal data obtained were used by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS) to define the classification and labelling (C&amp;L) for eye damage/irritation endpoint. However, the Draize test, a method which was never formally validated, has been widely criticized because of its technical limitations. In addition, ethical and economic issues and advances in scientific knowledge, and political and public pressures have made animal experimentation unsustainable. This scenario has consequently led to the development of nonanimal testing and protocols/approaches with considerable predictive value and relevance for humans. It is widely accepted that one single nonanimal method cannot cover all the criteria of damage/inflammation assessed by regulatory adopted in vivo animal testing. Thus, integrated testing strategies (ITS) amage (UN GHS Cat. 1) from materials that have reversible eye irritation effects (UN GHS Cat. 2).The biological functions of a cell may change in response to exposure to toxic agents. Toxicogenomics employs the recent developments in genomics, transcriptomics, and proteomics to study how a chemical impacts gene/protein expression and cell functions. https://www.selleckchem.com/products/abbv-744.html We describe a method for transcriptomic analysis by RNA sequencing based on Illumina HiSeq, NextSeq, or NovaSeq Systems followed by real-time qPCR validation. We also depict a method for proteomic analysis by "one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis" (1D SDS-PAGE) and a sample preparation procedure for "liquid chromatography in tandem with mass spectrometry" (LC-MS/MS), and we present some generic points to consider during LC-MS/MS.Freshwater planarians are free-living flatworms known for their regenerative ability. Being easily cultured under laboratory conditions, they are recognized test model organisms in regeneration, developmental biology, and neuropharmacological research. Also, they have been recently employed in toxicity testing where they displayed an array of sensitive and reliable responses to environmental stressors. Here, we outline simple and easy-to-follow protocols to evaluate effects of environmental contaminants and other stressors on survival, behavior (feeding and locomotor activity), and regeneration of freshwater planarians. These endpoints are comparable with responses of well-established ecotoxicological model species.Acetylcholinesterase (AChE) is a useful biomarker for organophosphate and carbamate pesticides exposure. The inhibition of this enzyme has been associated with neurotoxicity and alterations at higher levels of biological organization, such as behavior and development impairments. In this chapter, we describe the methodologies for analyses of AChE activity in pools of 96 h of embryos of zebrafish (Danio rerio) using a spectrophotometric method adapted to 96-well microtiter plates.The developing uterus is highly sensitive to a brief exposure to different substances, in particular those with endocrine-disrupting activity. Thus, exposure to environmental, nutritional, chemical, and other xenobiotic factors affecting signaling events during critical organizational periods can alter the normal course of uterine development with lasting consequences. In this chapter, we provide an experimental protocol to evaluate the development of the rat uterus as a toxicity biomarker at two different developmental time points (1) the neonatal period, on postnatal day (PND) 8, and (2) the prepubertal period, on PND21. In this experimental approach, we propose to assess (1) uterine morphology and cytodifferentiation, (2) uterine cell proliferation, and (3) the expression of proteins involved in uterine organogenetic differentiation. All these morphological and molecular markers are useful tools to determine the consequences of exposure to toxicants with the potential to disrupt the uterine development.The urinary bladder is a target organ of several toxic agents. Exposure to those agents induces mild-to-severe changes, which can be evaluated by different methods. Among them, the scanning-electron microscopy (SEM) is the "gold standard" for characterizing urothelial damage since it provides high-definition images, making it possible to detect early lesions on the surface of the urinary bladder. In addition, molecular technologies allow detecting changes in genetic material and investigating the interaction between genes and environmental stress in disease causation. The urinary bladder epithelium is where the most common type of bladder cancer occurs in humans, that is, the transitional-cell carcinoma (TCC). In animal models, the TCC can be similar to the disease in humans. Techniques to evaluate urothelium in experimental models aid in the comprehension of risk factors for urothelial carcinogenesis.